Currently, mainly four methods are performed in order to prepare a neural cell from an embryonic stem cell:    {circle around (1)} a method comprising treating a suspension culture of embryonic stem cell aggregates with retinoic acid, thereby inducing differentiation into various neural cells [Bain, G. et al, Dev. Biol., 168:642-657(1995)];    {circle around (2)} a method comprising preparing an embrioid body [referred to as EB], culturing the EB in a serum-free culture medium to obtain neural stem cells as nestin-positive cells, and culturing the neural stem cells in the presence of basic fibroblast growth factor (bFGF), to use the resulting cell in differentiation into neural cells [Okabe, S. et al., Mech. Dev., 59:89-102(1996)];    {circle around (3)} a method comprising culturing embryonic stem cells (ES cells) on established stroma cells, to form colonies, thereby differentiating the colonies into neural cells (SDIA method) [Kawasaki H. et al., Neuron, 28:31-40(2000)]; and    {circle around (4)} a method comprising carrying out suspension culture of ES cells in the presence of leukemia inhibiting factor (LIF), to prepare neural spheres from neural stem cells existed in ES cells in an amount of about 0.2% and then differentiating the neural spheres into neural cells [Tropepe V. et al, Neuron 30:65-78 (2001)].However, according to these methods, it is currently difficult to acquire sufficient amounts of cells for use in regenerative medicine from the viewpoint of teratogenicity caused by retinoic acid on differentiated cells, a time required for producing neural stem cells, the proportion of differentiation, an efficacy of yield and the like.
A neural stem cell, which is a cell having the multipotency into a neuron or a glial cell, and the self-renewal, plays a key role in transplantation regenerative medicine for the nervous system. As a method for maintaining and proliferating neural stem cells in an undifferentiated state, a neurosphere method has been established [Reynolds, B. A. et al., J. Neurosci., 12:4565-4574, (1992), Reynolds, B. A. et al., Science 255:1707-1710 (1992)]. According to the above-mentioned neurosphere method, when a cell population containing neural stem cells separated from the brain is cultured in DMEM: F-12 serum-free medium containing N2 supplement [insulin, transferrin, selenium and progesterone] and 20 ng/ml epidermal growth factor (referred to as EGF) and/or 20 ng/ml bFGF, only neural stem cells which can survive under the above-mentioned culture condition is proliferated. The proliferated neural stem cells are positive for a marker an intermediate filament nestin and form a cell aggregate (neurosphere), to become possible to be cultured in floating conditions. It has been found that when the above-mentioned neurosphere is cultured on a plate coated with adhesive substrate without growth factor, the cultured cells differentiate into neurons, astrocytes, oligodendrocytes or the like (multipotency). Further, it has been shown that when the neurosphere is dissociated into single cells and the resulting dissociated cells are cultured in a serum-free medium containing a growth factor, the dissociated cells form again a neurosphere (self-renewal). However, there are defects that the neurosphere is generated not from all of the cells but from a small portion of the total cell population.
In addition, as an alternative to the Neurosphere method, a monolayer culture method has been also established, wherein the method comprises culturing neural stem cells on a plate coated with an adhesive substrate, thereby proliferating and differentiating the cells. As the above-mentioned monolayer culture method, a method for performing monolayer culture comprising concentrating neural stem cells by a density gradient centrifugation [Palmer, T. D. et al., J. Neurosci., 19:8487-8597(1999)], or a method comprising performing monolayer culture of neural stem cells at a low density, thereby cloning and proliferating the cells [Ray, J. et al., Proc. Natl. Acad. Sci. USA, 90:3602-3606 (1993), Gage, F. H. et al., Proc. Natl. Acad. Sci. USA, 92:11879-11883 (1995)] is performed. According to the above-mentioned monolayer culture method, since each of cells can be identified, the method is suitable for cell lineage analysis of what sort of cells neural stem cells are differentiated into. However, the method is currently not suitable for the purpose of requiring a large amount of neural stem cells, since the neural stem cells are difficult to be maintained in the undifferentiated state and the proliferation of the neural stem cells is slow.
Therefore, in either of the Neurosphere method or the monolayer culture method, it is difficult to exactly determine which cells proliferate as neural stem cells in an undifferentiated state.